Type 1 fimbriae promote microbial adherence to a variety of mammalian cells and are thought to play an important role in the establishment of various infections. These type 1 fimbriae have mannose-specific adhesins, which enable these microorganisms to infect an organism. To facilitate the attachment to eukaryotic receptors, microorganisms bearing type 1 fimbriae assemble these fimbriae to present at their tips adhesin molecules (Hanson et al., 1998; Jones et al., 1995; Kuehn et al., 1992). Two major classes of fimbriae have been functionally defined in uropathogenic E. coli that can account for over 90% of the urinary tract infections. P fimbriae are expressed in about 70% of urinary tract isolates, particularly those from pyelonephritis patients, and they bind to the galactose-alpha(1,4)-galactose portion of the glycolipid receptors of the kidney (Johnson et al., 1992; O'Hanley et al., 1985; Stromberg et al., 1990; Stromberg et al., 1991). The type 1 fimbriae are expressed by over 90% of the uropathogenic E. coli, and they can bind, via mannose moieties, to the urothelial surface (Lin et al., 1995; Hutgren et al., 1985; O'Hanley et al., 1985; Sater et al., 1993; Schaeffer, 1991; Venegas et al., 1995). Immunohistochemical staining of voided urothelial cells of urinary tract infection patients showed adhering E. coli with type 1 fimbriae (Fujita et al., 1989; Kisielius et al., 1989).
Animal studies showed that E. coli expressing type 1 fimbriae, but not those harboring mutated ones, can cause urinary tract infection. These results clearly establish the functional importance of the mannose-sensitive, type 1 fimbriae in urinary tract infections (Iwahi et al., 1983; Keith et al., 1986; Schaeffer et al., 1987; Johanson et al., 1992). Virtually nothing is known, however, about the receptors that presumably bear the mannoses that are recognized by type 1 fimbriae. Consequently, the precise role of this kind of fimbriae and their functional relationship with the P fimbriae in various types of infections heretofore have not been well understood (Eisenstein, 1989; Falkow et al., 1992; Hopelmann and Tuamanen, 1992; Hultgren et al., 1993; Schoolnik et al., 1987; Stamm et al., 1989).
Many microorganisms express type 1 fimbriae, which accounts for their infectious nature. Among these microorganisms are Salmonella, Klebsiella, Citrobacter, Shigella, Enterobacter, Serratia, Proteus, Morganella, and Providencia.
Urinary tract infections are among the most common infectious diseases, accounting for almost five million cases annually and causing considerable morbidity and mortality (Schaeffer, 1994; Schoolnik, 1989; Stamm et al., 1989). Increasing incidence of antibiotics-resistant E. coli, which cause a great majority (up to 95%) of these infections, calls for additional therapeutic considerations. One useful approach entails the inhibition of bacterial attachment to the urothelial surface, which is a crucial initial event involving the precise interaction between a group of bacterial adhesive molecules, called adhesins, and their cognate urinary tract receptors (Falkow et al., 1992; Hopelman and Tuomanen, 1992; Hultgren et al., 1993; Schaeffer, 1988; Schoolnik et al., 1987; Stamm et al., 1989). Knowledge of the molecular details of the receptor:adhesin interface may provide a basis for rational drug design for preventing and treating a variety of infections.
Adhesion of causative bacteria to host urothelial surface depends on three principal factors: the expression of proper bacterial adhesins, the availability of host receptors, and the functional status of an innate host defense mechanism.
Pharmaceutical agents which prevent attachment of bacterial pathogens to particular receptors on the host cells' membranes would cause little interference with normal host function, since they would act only on the attachment of the pathogens to receptors. The bacteria would survive and continue to grow, but would be more susceptible to elimination by host defenses including the flushing action produced by urine flow. Counteraction of infection by interfering with or preventing attachment of type 1 fimbriae to host cells' membranes would be less likely to result in selection for drug resistance because the anti-adhesin compounds involved need not favor the growth of mutants that are resistant to their action. In other words, compounds which interfere with or prevent attachment to host cells' receptors do not affect bacterial growth, so there is no favoring of mutants which are resistant to the action of these compounds.
Asymmetric unit membrane (AUM), a highly specialized membrane, covers the bulk of the urinary tract and performs two functions: it serves as a physically stable and yet flexible permeability barrier against urine and provides a vehicle for the reversible adjustment of the bladder surface area by incorporating AUM-containing cytoplasmic vesicles to the luminal surface during bladder expansion and by retrieving the AUM back into the cytoplasm during bladder contractions. However, how AUM performs these functions is unclear.
Significant progress has recently been made to characterize biochemically the apical surface of mammalian urothelium, which is covered with numerous rigid-appearing 0.3-0.5 micron plaques. In cross-sections, the luminal leaflet of the plaque membrane is twice as thick as the cytoplasmic leaflet, hence the term "asymmetrical unit membrane". Urothelial asymmetrical unit membranes have recently been found to contain four major integral membrane proteins, which have been named uroplakin Ia, 27 kDa; uroplakin Ib, 28 kDa; uroplakin II, 15 kDa; and uroplakin III, 47 kDa. (Lin et al., 1994; Lin et al., 1995; Walz et al., 1995; Wu et al., 1994; Wu et al., 1990; Wu and Sun, 1993; Yu et al., 1994; Yu et al., 1990).
All of the major asymmetrical unit membrane proteins have dominant luminal domains with relatively little or, in the cases of uroplakin I proteins and uroplakin II, almost no cytoplasmic domains. The asymmetrical distribution of the uroplakin domains across the lipid bilayer suggests that the luminal domains may interact to form the 16-nm protein particles protruding luminally and may explain why the luminal leaflet is thicker than its cytoplasmic leaflet. Ultrastructural localization confirmed that the uroplakins are associated with the asymmetrical unit membrane plaques in situ. Because these plaques occupy 70-80% of the urothelial apical surface and are only interrupted by short interplaque "hinge" areas, these four uroplakins, as the major asymmetrical unit membrane subunits, are the predominant protein components of the urothelial apical surface.
Together, the four uroplakins form 16-nm luminal protein particles that are arranged in two-dimensional crystalline arrays. Image processing revealed that each 16-nm particle consists of six inner and six outer domains interconnected, forming a continuous strand in the shape of a twisted ribbon (Waltz et al., JMB, 1995). cDNA cloning showed that uroplakins Ia and Ib are closely related isoforms, sharing 39% amino acid sequences; they belong to a family of cell surface proteins having four conserved transmembrane domains (Yu et al., JCB, 1995). Uroplakins II (UPII) and III (UPIII) have a single transmembrane domain located near the C-terminus (Wu, JCS, 1994; Lin, JBC, 1995). In a nearest-neighbor analysis using chemical cross linking, it has been demonstrated that uroplakins Ia (UPIa) and Ib (UPIb) can be cross-linked to uroplakins II and III, respectively, raising the possibility that two types of 16-nm particles exist and that each contains two related pairs of uroplakins (Wu, JBC, 1995).
Although it has long been hypothesized that type 1 fimbriated microorganisms bind to the urothelial surface and that this binding plays a major role in infections such as urinary tract infections, the receptors have not heretofore been identified. Consequently, little is known about the details of this bacterial fimbriae:urothelial receptor binding. Also, it was impossible to design simple and physiologically relevant screening for drugs that can interfere with this binding. Identification of urothelial receptors will solve these problems.
While yeast or intestinal epithelial cells have been traditionally used to screen drugs for efficacy in treating urinary tract infections, there is no assurance that bacterial adhesion to yeast or to intestinal epithelial cells is the same as bacterial adhesion to epithelial cells in the urinary tract. It is not known if the type 1 fimbriated microorganisms bind to a protein backbone as well as to mannose, which means that yeast or epithelial cells may not be as specific with respect to microbial adhesion in screening drugs as would be the actual urothelial receptors.
Although mannose was traditionally regarded as the sole binding site for type 1 fimbriae, several recent studies revealed the complexity of the binding of type 1 fimbriae to its receptors. Sokurenko and coworkers (1992) identified three types of adhesive properties based on the binding of type-1 fimbriated E. coli strains to different substrates including mannan, fibronectin, deglycosylated fibronectin and a synthetic peptide corresponding to the N-terminus of fibronectin. Surprisingly, several type 1-fimbriated strains, both clinical and laboratory, bound to deglycosylated fibronectin or even the synthetic peptide, in a mannose-sensitive fashion, suggesting the possible binding to a protein backbone (Sokurenko et al., 1992). In another recent study, Sokurenko et al. (1995) showed that type 1-fimbriated E. coli isolates from urinary tract infections had a much higher affinity to immobilized mannan than a group of fecal E. coli isolates, suggesting a hitherto unsuspected binding specificity between certain E. coli strains and urothelial receptors (Sokurenko et al., 1995). Collectively, these findings strongly suggest that old systems that involve the use of non-urothelial material in studying pathogenesis and drug screening may not be directly relevant for infection by type 1 fimbriated microorganisms. Thus, previous drug screening methods which relied on yeast may not be accurate in detecting which compounds actually inhibit infection, i.e., binding, by type 1-fimbriated microorganisms.
Traditional methods for drug screening have relied upon structure-activity studies and incremental improvement of drug leads by evaluating analogues produced by medicinal chemistry. This is a time-consuming approach that may well overlook many compounds which have completely novel structures.
To facilitate screening large numbers of test samples, which need not be related compounds, a robotic system has recently become available (Heguey et al, 1995). This robotic system enables complete automation of every step in the drug screening process. On-line cell incubation facilities interface with liquid handling systems for diluting and adding test samples, and other units are robotically manipulated in an assay loop that transfers microplates between the plate washed, the reagent addition works station, and a 96-well luminometer. Two robotic arm assemblies are used in each system. Data are captured automatically into a processing network that performs quality control evaluations on each individual microplate assay, as well as rapid data reduction and analysis. Each robotic system can assay up to 10,000 compounds per week. By using multiple targets in the primary screen, efficacy, cytotoxicity, and initial specificity are evaluated rapidly. Compounds of interest identified by the primary screen are then further evaluated in secondary screens and, if necessary, tertiary assays. Animal models are then employed for the final stages of drug development (Heguey et al., 1995).